LprG as a chaperone of immune adjuvants

ABSTRACT

An adjuvant combination that stimulates immune activation or response includes a hydrophobic immune adjuvant and a pathogen derived lipoprotein that chaperones the hydrophobic immune adjuvant to an immune receptor.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.61/147,304, filed Jan. 26, 2009, the subject matter, which isincorporated herein by reference.

BACKGROUND

Immune adjuvants are molecules that signal through receptors to enhanceimmune responses. One example is the addition of adjuvants to vaccinesto enhance the response to the vaccine antigens. The improvement ofvaccine adjuvants is an important current goal in vaccine development.Adjuvants may also be important for other types of immunotherapy,including potential treatments under development for cancer,autoimmunity and other disorders in which the immune system may play arole in either pathophysiology or therapy.

Immune adjuvants may be derived from microbial molecules that generateresponses in the mammalian recipient. Immune adjuvants are recognized byreceptors, often in the category of innate immune receptors. One exampleof an innate immune receptor is the Toll-like receptor (TLR) family.TLRs recognize acylated molecules, such as lipoproteins, lipopeptidesand glycolipids. TLRs can also respond to agonists by signaling toinduce immune activation.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to adjuvant combinations that stimulateimmune activation. The adjuvant combinations include a hydrophobicimmune adjuvant and a pathogen derived lipoprotein that chaperones thehydrophobic immune adjuvant to an immune receptor. In one aspect of theinvention, the pathogen derived lipoprotein includes LprG.

In another aspect of the invention, the hydrophobic immune adjuvant is aglycolipid immune adjuvant. The hydrophobic immune adjuvant can alsoinclude at least one of a TLR agonist or TLR ligand. By way of example,the hydrophobic immune adjuvant can be at least one of a TLR2 agonist orTLR4 agonist.

The present invention also relates to an immune stimulating complex thatincludes a hydrophobic immune adjuvant and LprG. The LprG can be presentin an amount effective to chaperone the immune adjuvant to an immunereceptor. The hydrophobic immune adjuvant can be a glycolipid immuneadjuvant. The hydrophobic immune adjuvant can also include at least oneof a TLR2 agonist or TLR4 agonist.

The present invention further relates to a vaccine that includes avaccine antigen, a hydrophobic TLR agonist, and LprG. The LprG can beprovided in the vaccine in an amount effective to chaperone thehydrophobic TLR agonist to a TLR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates NA-LprG carries a mycobacterial TLR2 agonist. (A)HEK293.TLR2 cells show a dose-dependent IL-8 response to LprA, LprG, andNA-LprG, but no response to NA-LprA. Top panel, bioassay withHEK293.TLR2 cells. Bottom panel, bioassay with HEK293.TLR2-CD14 cells.Control HEK293 cells lacking TLR2 and CD14 failed to respond to all fourproteins, similar to the response seen to NA-LprA in these panels (datanot shown). (B) Alignment of primary amino acid sequences of LprA (SEQID NO: 1) and LprG (SEQ ID NO: 2). Shared residues are highlighted. (C,D) TLR2 activity of NA-LprG/NA-LprA chimeric molecules. HEK293.TLR2cells were incubated for 12 h with NA-LprG, NA-LprA or chimeric proteinscombining the N-terminal half (NTD) and C-terminal half (CTD) of NA-LprGand NA-LprA. (E, F) NA-LprG can acquire TLR2 agonist activity frommycobacterial lysates. NA-LprG and NA-LprA were expressed in E. coli,purified, purified by Ni-affinity and ion exchange chromatography,incubated with control buffer or a lysate of M. smegmatis (E) or MtbH37Ra (F), repurified by Ni-affinity and ion exchange chromatography,and incubated with HEK293.TLR2 cells for 12 h. For all data panels, IL-8production was quantified by ELISA, and data are reported as themean+/−SD of triplicate HEK293.TLR2 assays from a representative of atleast 3 independent experiments.

FIG. 2 illustrates crystal structures of NA-LprG reveals a hydrophobicpocket with the potential to carry a TLR2 agonist.

FIG. 3 illustrates plots showing site-directed mutagenesis and singleamino acid alteration of the hydrophobic pocket reduces the ability ofNA-LprG to bind and deliver TLR2 agonists. (A) TLR2 Activity of NA-LprGand NA-LprG-V91W expressed in M. smegmatis and tested on HEK293.TLR2cells as in FIG. 1. (B) TLR2 activity of NA-LprG and NA-LprG-V91Wexpressed in E. coli, purified, incubated with Mtb lysate and repurifiedas in FIG. 1F. Data for panels A and B are reported as the mean+/−SD oftriplicate HEK293.TLR2 assays from a representative of at least 3independent experiments.

FIG. 4 illustrates plots showing NA-LprG activity is dependent on TLR1and TLR2. (A, B) Macrophage response to NA-LprG is dependent on TLR2 andTLR1, but not dependent on TLR6. NA-LprG was purified from M. smegmatis(panel A) or purified from E. coli, incubated with Mtb H37Ra lysate andthen repurified (panel B). NA-LprG preparations were incubated for withbone marrow-derived macrophages from TLR2−/−, TLR1−/−, TLR6−/− orwild-type mice, and TNF-alpha production was determined by ELISA. (C-D)Macrophage responses to mycobacterial glycolipids are dependent on TLR2and TLR1, but not dependent on TLR6. TLR dependency was assessed asabove. (E, F). TLR2 responses to triacylated and diacylated lipopeptidesare dependent on TLR1 and TLR6, respectively. (G) Response to non-TLR2signaling (by CpG ODN 1826 TLR9 agonist) is intact in knockout celllines. Data are reported as the mean+/−SD of triplicate macrophageassays from a representative of at least 3 independent experiments.

FIG. 5 illustrates immunoblots showing Mycobacterial glycolipids areassociated with NA-LprG. SDS-PAGE analysis of proteins purified from M.smegmatis. Samples were visualized by silver stain (A, left panel),Pro-Q stain for carbohydrates (A, right panel), polyclonal anti-BCGWestern blot (B) and anti-His₆ Western blot (C).

FIG. 6 illustrates triacylated Ac₁PIM₂ is specifically associated withLprG. Nanospray ionization mass spectrometry in negative mode was usedto analyze molecules associated with NA-LprG (A), NA-LprG-V91W (B) andNA-LprA (C).

FIG. 7 illustrates plots showing NA-LprG binds purified mycobacterialglycolipids and facilitates their recognition by TLR2. NA-LprG waspurified from E. coli, incubated with preparations of PIM (containing amixture of PIM₁/PIM₂ or PIM₁/PIM₂/PIM₆), LM from M. smegmatis, PI-LAMfrom M. smegmatis (Ms-LM and Ms-LAM, respectively), LM from Mtb H37Rvand ManLAM purified from Mtb H37Rv. NA-LprG was then repurified andassessed for TLR2 activity using HEK293.TLR2-CD14 cells as in FIG. 1.Data are reported as the mean+/−SD of triplicate HEK293.TLR2-CD14 assaysfrom a representative of at least 3 independent experiments.

FIG. 8 illustrates plots showing the glycolipid binding site contributessignificantly to the total TLR2 agonist activity of acylated LprG.HEK.293.TLR2-CD14 cells were used to assess TLR2 agonist activity ofacylated LprG and LprG-V91W as in FIG. 1. Data are reported as themean+/−SD of triplicate HEK293.TLR2-CD14 assays from a representative ofat least 3 independent experiments.

DETAILED DESCRIPTION

The present invention relates to pathogen derived lipoproteinformulations (e.g., LprG formulations) that unexpectedly can be used asefficient delivery vehicles for hydrophobic immune adjuvants, such ashydrophobic TLR ligands. It was found that pathogen derived lipoproteinformulations are a particularly effective vehicle for delivery ofhydrophobic immune adjuvants, such as TLR ligands, particularly thosethat would be immunologically inert or poorly immunostimulatory if notadministered together with the pathogen derived lipoproteinformulations. Although not intending to be bound by any particularmechanism, it is postulated that the pathogen derived lipoproteinformulations enhance delivery of such ligands to their respectivereceptors (e.g., particular TLR family members) by complexing with orbinding to the hydrophobic immune adjuvants and chaperoning the immuneadjuvant to the desired immune receptor. This has resulted in theobserved synergistic enhancement of innate immune responses when theligand/lipoprotein formulation is used in particular experimentaltherapeutic settings.

It was unexpected that use of pathogen derived lipoprotein formulationsof the present invention could essentially chaperonepreviously-characterized immunologically substantially inert hydrophobicTLR ligands to TLR to stimulate an immune response. This observationbroadens the genus of TLR ligands that can be used for immunostimulatorypurposes to include TLR ligands with no previously characterizedimmunostimulatory motif and/or no or low previously characterizedimmunostimulatory potential.

These findings indicate that adjuvant combinations comprising pathogenderived lipoprotein formulations and hydrophobic immune adjuvants areuseful in optimizing innate immune therapies, such as but not limited tothose directed to infectious disease, cancers, allergy and asthma.

In one aspect of the invention, the pathogen derived lipoprotein caninclude an LprG lipoprotein that is derived from Mycobacteriumtuberculosis (Mtb) and that can chaperone a hydrophobic immune adjuvantto an immune receptor. It was found that acylated LprG and non-acylatedLprG can act as a myobacterial glycolipid chaperone and deliver theglycopids for recognition by TLRs (e.g., TLR-2).

In another aspect of the invention, the hydrophobic immune adjuvant caninclude a glycolipid adjuvant, such as a pathogen-derived (e.g.,Myobacterium) glycolipid adjuvant. In general terms, an adjuvant is asubstance that non-specifically enhances the immune response to anantigen, i.e., is an immunostimulant. In general terms, a glycolipid isa cell membrane lipid molecule with a carbohydrate chain attached to ahydrophobic tail. Glycolipid adjuvants of the present invention caninclude modified lipopolysaccharides. The lipopolysaccharide is modifiedsuch that its toxicity is reduced compared to the corresponding wildtype lipopolysaccharide or lipopolysaccharide from which it has beenderived.

In one example, the glycolipid immune adjuvant can be a hydrophobic TLRligand and/or a hydrophobic TLR agonist. The hydrophobic TLR ligandand/or hydrophobic TLR agonist can be formulated with the pathogenderived lipoprotein in any number of ways. For example, the TLR ligandand/or TLR agonist can simply be mixed with the pathogen derivedlipoproteins.

As used herein, a TLR ligand is a molecule that binds to a TLR (i.e., aToll-like receptor). There are a number of TLR identified to dateincluding TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10and TLR11. There are similarly a number of TLR ligands identified todate, some of which have been observed to be immunostimulatory (e.g.,CpG oligonucleotides). The invention intends to embrace hydrophobic TLRligands that have been previously identified as being TLR ligands andwhich may have also been observed to be immunologically inert. As usedherein, an immunologically inert TLR ligand is one which has beenobserved to have no or low immunostimulatory potential. The inventionalso intends to embrace compounds that according to the invention aretested in the presence and absence of a pathogen derived lipoprotein(e.g., LprG) and found to be transformed from an inert compound to animmunostimulatory compound.

Screening assays for TLR ligands have been described in, for example,U.S. Patent Application Publication No. US 2003/0104523, published Jun.5, 2003, which is incorporated herein in their entirety. The inventionintends to embrace the use of compounds that are shown to be TLR ligands(e.g., via radiolabeled ligand-receptor assays) but which when comparedto, for example, immunostimulatory adjuvants appear to be inert becausetheir relative immunostimulatory potential is negligible ortherapeutically non-useful in comparison.

The adjuvant combinations comprising the pathogen derived lipoproteinand hydrophobic immune adjuvants can further include an antigen forinducing an immunological response in a mammal (e.g., human). Theantigen can be provided with the adjuvant combinations in a vaccine thatcan be inoculated in an individual to produce antibody, preferably IgA,and/or a T cell immune response. The response can be adequate to protectsaid individual from infection, particularly bacterial or viralinfection. Thus, the immunological response may be used therapeuticallyor prophylatically.

The vaccine antigen can include but is not limited to bacterial, viral,parasitic, allergens, autoantigens and tumor associated antigens.Particularly, the antigen can include protein antigens, peptides, wholeinactivated organisms, and the like.

Specific examples of antigens that can be used in the invention includeantigens from hepatitis A, B, C or D, influenza virus, Listeria,Clostridium botulinum, tuberculosis, tularemia, Variola major(smallpox), viral hemorrhagic fevers, Yersinia pestis (plague), HIV,herpes, pappilloma virus, and other antigens associated with infectiousagents. Other antigens include antigens associated with a tumor cell,antigens associated with autoimmune conditions, allergy and asthma.Administration of such an antigen in conjunction with the subject immunecombination can be used in a therapeutic or prophylactic vaccine forconferring immunity against such disease conditions.

In some embodiments, the methods and compositions can be used to treatan individual at risk of having an infection or has an infection byincluding an antigen from the infectious agent. An infection refers to adisease or condition attributable to the presence in the host of aforeign organism or an agent, which reproduce within the host. A subjectat risk of having an infection is a subject that is predisposed todevelop an infection. Such an individual can include for example asubject with a known or suspected exposure to an infectious organism oragent. A subject at risk of having an infection can also include asubject with a condition associated with impaired ability to mount animmune response to an infectious agent or organism, for example asubject with a congenital or acquired immunodeficiency, a subjectundergoing radiation or chemotherapy, a subject with a burn injury, asubject with a traumatic injury, a subject undergoing surgery, or otherinvasive medical or dental procedure, or similarly immunocompromisedindividual.

Infections which may be treated or prevented with the vaccinecompositions of this invention include bacterial, viral, fungal, andparasitic. Other less common types of infection also include arerickettsiae, mycoplasms, and agents causing scrapie, bovine spongiformencephalopathy (BSE), and prion diseases (e.g., kuru andCreutzfeldt-Jacob disease). Examples of bacteria, viruses, fungi, andparasites that infect humans are well know. An infection may be acute,subacute, chronic or latent and it may be localized or systemic.Furthermore, the infection can be predominantly intracellular orextracellular during at least one phase of the infectious organism'sagent's life cycle in the host.

Bacteria infections against which the subject vaccines and methods maybe used include both Gram negative and Gram positive bacteria. Examplesof Gram positive bacteria include but are not limited to Pasteurellaspecies, Staphylococci species, and Streptococci species. Examples ofGram negative bacteria include but are not limited to Escherichia coli,Pseudomonas species, and Salmonella species. Specific examples ofinfectious bacteria include but are not limited to Heliobacter pyloris,Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (forexample M. tuberculosis, M. avium, M. intracellilare, M. kansaii, M.gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseriameningitidis, Listeria monocytogeners, Streptococcus pyogenes, (group AStreptococcus), Streptococcus agalactiae (Group B Streptococcus),Streptococcus (viridans group), Streptococcus faecalis, streptococcusbovis, Streptococcus (anaerobic spp.), Streptococcus pneumoniae,pathogenic Campylobacter spp., Enterococcus spp., Haemophilusinfluenzae, Bacillus anthracis, Corynebacterium diptheriae,Corynebacterium spp., Erysipelothrix rhusiopathie, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasteurella multocida, Bacteroides spp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelii.

Examples of viruses that cause infections in humans include but are notlimited to Retroviridae (for example human deficiency viruses, such asHIV-1 (also referred to as HTLV-III), HIV-II, LAC or IDLV-III (LAV orHIV-III and other isolates such as HIV-LP, Picornaviridae (for examplepoliovirus, hepatitis A, enteroviruses, human Coxsackie viruses,rhinoviruses, echoviruses), Calciviridae (for example strains that causegastroenteritis), Togaviridae (for example equine encephalitis viruses,rubella viruses), Flaviviridae (for example dengue viruses, encephalitisviruses, yellow fever viruses) Coronaviridae (for examplecoronaviruses), Rhabdoviridae (for example vesicular stomata viruses,rabies viruses), Filoviridae (for example Ebola viruses) Paramyxoviridae(for example parainfluenza viruses, mumps viruses, measles virus,respiratory syncytial virus), Orthomyxoviridae (for example influenzaviruses), Bungaviridae (for example Hataan viruses, bunga viruses,phleoboviruses, and Nairo viruses), Arena viridae (hemorrhagic feverviruses), Reoviridae (for example reoviruses, orbiviruses, rotaviruses),Bimaviridae, Hepadnaviridae (hepatitis B virus), Parvoviridae(parvoviruses), Papovaviridae (papilloma viruses, polyoma viruses),Adenoviridae (adenoviruses), Herpeviridae (for example herpes simplexvirus (HSV) I and II, varicella zoster virus, pox viruses) andIridoviridae (for example African swine fever virus) and unclassifiedviruses (for example the etiologic agents of Spongiformencephalopathies, the agent of delta hepatitis, the agents of non-A,non-B hepatitis (class 1 enterally transmitted; class 2 parenterallytransmitted such as Hepatitis C); Norwalk and related viruses andastroviruses).

Examples of fungi include Aspergillus spp., Coccidoides immitis,Cryptococcus neoformans, Candida albicans and other Candida spp.,Blastomyces dermatidis, Histoplasma capsulatum, Chlamydia trachomatis,Nocardia spp., and Pneumocytis carinii.

Parasites include but are not limited to blood-borne and/or tissueparasites such as Babesia microti, Babesi divergans, Entomoebahistolytica, Giarda lamblia, Leishmania tropica, Leishmania spp.,Leishmania braziliensis, Leishmania donovdni, Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Toxoplasmagondii, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagus' disease) and Toxoplasmagondii, flat worms, and round worms.

The present invention further embraces the use of the subject adjuvantcombinations in treating proliferative diseases, such as cancers. Canceris a condition of uncontrolled growth of cells, which interferes withthe normal functioning of bodily organs and systems. A subject that hasa cancer is a subject having objectively measurable cancer cells presentin the subjects' body. A subject at risk of developing cancer is asubject predisposed to develop a cancer, for example based on familyhistory, genetic predisposition, subject exposed to radiation or othercancer-causing agent. The adjuvant combinations and compositionsaccording to the invention can be used to treat a variety of cancers orsubjects at risk of developing cancer, by the inclusion of atumor-associated-antigen (TAA). Examples of such cancers include breast,prostate, colon, blood cancers such as leukemia, chronic lymphocyticleukemia, and the like. The vaccination methods of the invention can beused to stimulate an immune response to treat a tumor by inhibiting orslowing the growth of the tumor or decreasing the size of the tumor. Atumor associated antigen can also be an antigen expressed predominantlyby tumor cells but not exclusively.

Additional cancers include but are not limited to basal cell carcinoma,biliary tract cancer, bladder cancer, bone cancer, brain and centralnervous system (CNS) cancer, cervical cancer, choriocarcinoma,colorectal cancers, connective tissue cancer, cancer of the digestivesystem, endometrial cancer, esophageal cancer, eye cancer, head and neckcancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynxcancer, liver cancer, lung cancer (small cell, large cell), lymphomaincluding Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma;neuroblastoma; oral cavity cancer (for example 11p, tongue, mouth andpharynx); ovarian cancer; pancreatic cancer; retinoblastoma;rhabdomyosarcoma; rectal cancer; cancer of the respiratory system;sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer;uterine cancer; cancer of the urinary system; as well as othercarcinomas and sarcomas.

The adjuvant combinations and compositions containing according to theinvention can also be used to treat autoimmune diseases such as multiplesclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis or otherautoimmune disorders. Other autoimmune disease which potentially may betreated with the vaccines and immune adjuvants of the invention includeCrohn's disease and other inflammatory bowel diseases such as ulcerativecolitis, systemic lupus eythematosus (SLE), autoimmuneencephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis,Goodpasture's syndrome, pemphigus, Graves disease, autoimmune hemolyticanemia, autoimmune thrombocytopenic purpura, scleroderma withanti-collagen antibodies, mixed connective tissue disease, polypyositis,pernicious anemia, idiopathic Addison's disease, autoimmune associatedinfertility, glomerulonephritis) for example crescenticglomerulonephritis, proliferative glomerulonephritis), bullouspemphigoid, Sjogren's syndrome, psoriatic arthritis, insulin resistance,autoimmune diabetes mellitus (type 1 diabetes mellitus; insulindependent diabetes mellitus), autoimmune hepatitis, autoimmunehemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmunehepatitis, autoimmune hemophilia, autoimmune lymphoproliferativesyndrome, autoimmune uveoretinitis, and Guillain-Bare syndrome.Recently, arteriosclerosis and Alzheimer's disease have been recognizedas autoimmune diseases. Thus, in this embodiment of the invention theantigen will be a self-antigen against which the host elicits anunwanted immune response that contributes to tissue destruction and thedamage of normal tissues.

The adjuvant combinations and compositions containing according to theinvention can also be used to treat asthma and allergic and inflammatorydiseases. Asthma is a disorder of the respiratory system characterizedby inflammation and narrowing of the airways and increased reactivity ofthe airways to inhaled agents. Asthma is frequently although notexclusively associated with atopic or allergic symptoms. Allergy isacquired hypersensitivity to a substance (allergen). Allergic conditionsinclude eczema, allergic rhinitis, or coryza, hay fever, bronchialasthma, urticaria, and food allergies and other atopic conditions. Anallergen is a substance that can induce an allergic or asthmaticresponse in a susceptible subject. There are numerous allergensincluding pollens, insect venoms, animal dander, dust, fungal spores,and drugs.

Examples of natural and plant allergens include proteins specific to thefollowing genera: Canine, Dermatophagoides, Felis, Ambrosia, Lotium,Cryptomeria, Alternaria, Alder, Alinus, Betula, Quercus, Olea,Artemisia, Plantago, Parietaria, Blatella, Apis, Cupressus, Juniperus,Thuya, Chamaecyparis, Periplanet, Agopyron, Secale, Triticum, Dactylis,Festuca, Poa, Avena, Holcus, Anthoxanthum, Arrhenatherum, Agrostis,Phleum, Phalaris, Paspalum, Sorghum, and Bromis.

It is understood that the adjuvant combinations and compositionscontaining according to the invention can be combined with othertherapies for treating the specific condition, e.g., infectious disease,cancer or autoimmune condition. For example in the case of cancer theinventive methods may be combined with chemotherapy or radiotherapy.

The adjuvant combinations of the invention can be administered locallyor systemically by any method known in the art including but not limitedto intramuscular, intravenous, intradermal, subcutaneous,intraperitoneal, intranasal, oral or other mucosal routes. Additionalroutes include intracranial (for example intracisternal, orintraventricular), intraorbital, ophthalmic, intracapsular, intraspinal,and topical administration. The adjuvants and vaccine compositions ofthe invention can be administered in a suitable, nontoxic pharmaceuticalcarrier, or can be formulated in microcapsules or a sustained releaseimplant. The immunogenic compositions of the invention can beadministered multiple times, if desired, in order to sustain the desiredcellular immune response. The appropriate route, formulation, andimmunization schedule can be determined by one skilled in the art.

The subject adjuvant combinations can be administered with aphysiologically acceptable carrier such as physiological saline. Thecomposition may also include another carrier or excipient such asbuffers, such as citrate, phosphate, acetate, and bicarbonate, aminoacids, urea, alcohols, ascorbic acid, phospholipids, proteins such asserum albumin, ethylenediamine tetraacetic acid, sodium chloride orother salts, liposomes, mannitol, sorbitol, glycerol and the like. Theadjuvants of the invention can be formulated in various ways, accordingto the corresponding route of administration. For example, liquidformulations can be made for ingestion or injection, gels or procedurescan be made for ingestion, inhalation, or topical application. Methodsfor making such formulations are well known and can be found in forexample, “Remington's Pharmaceutical Sciences,” 18.sup.th Ed., MackPublishing Company, Easton Pa.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein. Thevarious references to journals, patents, and other publications whichare cited herein comprise the state of the art and are incorporated byreference as though fully set forth.

EXAMPLES

To investigate the roles of acyl chains and other potential determinantsin TLR2 agonist activity of Mtb lipoproteins, we expressed nonacylated(NA) variants of LprA and LprG, termed NA-LprA and NA-LprG,respectively. While NA-LprA appeared to lose its TLR2 stimulatoryactivity, NA-LprG retained TLR2 stimulatory capacity. The crystalstructure of NA-LprG revealed a putative binding pocket lined withhydrophobic residues, and biochemical studies revealed binding oftriacylated Mtb glycolipids to NA-LprG. Mutation of the putative bindingpocket reduced TLR2 agonist activity of acylated LprG and selectivelyabolished the ability of NA-LprG to bind triacylated phospholipids.These results present two new interpretations. First, we show that theevolutionary function of LprG in mycobacteria is to act as a glycolipidchaperone to mediate trafficking and delivery of glycolipid duringconstruction of the mycobacterial envelope, contributing to virulenceand providing potential opportunities for targeting in drug design.Second, we show that a glycolipid chaperone activity of LprG facilitatesrecognition of triacylated glycolipids by TLR2. This presents a novelparadigm for recognition of hydrophobic TLR2 agonists that requirechaperones for delivery to TLR2; host cells may co-opt the function ofmicrobial chaperones, providing a mechanism to enhance innate immunerecognition of Mtb or other bacteria that express hydrophobic TLR2agonists.

Having previously determined that purified wild-type LprG and LprAproteins activate TLR2, we sought to determine whether the mechanisminvolved triacylation at the N-terminal cysteine. Since acylation ofthese lipoproteins is mediated by a common enzymatic pathway, weinvestigated whether the protein structure of these lipoproteins couldcontribute to TLR2 agonist activity independent of the presence of theN-terminal cysteine required for tri-acylation. Recombinant 6×His-taggedacylated and non-acylated versions of LprA and LprG were expressed andassessed for TLR2 activity (non-acylated forms were expressed withoutthe leader peptide and with the acylated N-terminal cysteine replacedwith methionine). Mtb LprG was a more potent TLR2 agonist than ahomologous Mtb lipoprotein, LprA, in a bioassay with TLR2-transfectedHEK293.TLR2 cells. NA-LprA did not induce TLR2 responses implicating thetri-acyl motif in its mechanisms of action. However, NA-LprG retainedsignificant TLR2 agonist activity (lower than acylated LprG but as highor higher than acylated LprA) (FIG. 1A). Interestingly, expression ofCD14 (in HEK293.TLR2-CD14 cells) enhanced the apparent potency ofNA-LprG but not acylated LprG or LprA (FIG. 1A), consistent with otherevidence that the mechanism for recognition of the NA-LprG-associatedTLR2 agonist activity was different than for acylated LprG and LprA(below). These data indicate that determinants other than acylation ofthe N-terminal cysteine contribute to TLR2 agonist activity of LprG.

To test whether specific peptide sequences or domains were required forTLR2 agonist activity of NA-LprG, we produced N-terminal truncationmutants of NA-LprG. Truncations within the first 14 amino acids did notaffect TLR2 agonist activity, and further truncation involving the firstalpha helix by subsequent structural studies, produced instability andpoor expression (data not shown). Since LprG and LprA are homologous(FIG. 1B), and LprA lacks acyl-independent TLR2 activity, an alternativestrategy was to produce full-length chimeric molecules containingdifferent portions of LprG and LprA to determine which portions of LprGconferred TLR2 activity. Chimeric molecules were produced containing theN-terminal half of NA-LprA and the C-terminal half of NA-LprG, or viceversa, and tested for TLR2 agonist activity with HEK293.TLR2 cells (FIG.1C, D). Both chimeric molecules were active, although greater activitywas associated with LprG sequence in the C-terminal domain, suggestingthat both halves of the molecule contributed to agonism, perhaps througha conformational determinant.

Since a closely related lipoprotein, LppX, is thought to bind andtransport hydrophobic phthiocerol dimycocerosates, we hypothesized thatLprG might non-covalently bind a lipid TLR2 agonist via aconformation-dependent binding site. This hypothesis was supported bythe observation that NA-LprG had reduced activity when expressed in E.coli instead of M. smegmatis (FIG. 1E), suggesting that TLR2 agonist(s)carried by LprG may be specific to mycobacteria. Further suggestingnon-covalent capture of a cell wall product, the activity of NA-LprGpurified from E. coli was significantly increased following incubationwith lysates of either M. smegmatis (FIG. 1E) or Mtb H37Ra (FIG. 1F).Similar charging of TLR2 agonist activity of NA-LprG from E. coli wasobserved with lysate from Mtb H37Rv demonstrating that the effect isrelevant to virulent Mtb. These results suggest that NA-LprG bindsmycobacterial TLR2 agonist(s) and delivers them for recognition by TLR2.

To test the hypothesis that the hydrophobic pocket of LprG serves as abinding site, we performed site-directed mutagenesis to create a V91Wmutant, replacing a valine lining the interior pore with a bulkytryptophan. NA-LprG-V91W was stably expressed in M. smegmatis, and itsability to activate TLR2 was significantly reduced relative to NA-LprGexpressed in M. smegmatis (FIG. 3) Mutations at other sites (V194R andV217F) also decreased TLR2 agonist activity of NA-LprG (data not shown).Furthermore, when NA-LprG-V91W was expressed in E. coli, purified andthen incubated with a sonicate of M. smegmatis or Mtb, the V91W mutantlacked the ability to acquire TLR2 agonist activity from mycobacteria(FIG. 3) and data not shown). These results provide strong support forthe hypothesis that the hydrophobic cavity serves as a binding site forTLR2 agonist(s), which are then delivered by LprG for recognition byTLR2.

Since TLR2 co-receptor dependence varies with different types of TLR2agonists, we investigated properties of TLR2 agonist(s) putativelyassociated with NA-LprG by testing the dependence of NA-LprG signalingon TLR2 co-receptors, TLR1 and TLR6. NA-LprG from M. smegmatis wasincubated with bone marrow-derived macrophages from mice geneticallydeficient in TLR1, TLR2 or TLR6, and TNFα production was quantified byELISA. Recognition of NA-LprG or associated molecules was deficient inTLR2^(−/−) and TLR1^(−/−) macrophages but not TLR6^(−/−) macrophages(FIG. 4A). Similar results were observed with NA-LprG that was expressedin E. coli and then incubated with Mtb lysate to allow loading ofNA-LprG with Mtb-derived TLR2 agonist(s) (FIG. 4B). These resultssuggest that the activity associated with NA-LprG signals throughTLR2/TLR1 heterodimers, similar to the mycobacterial glycolipids LAM(FIG. 4C), LM (FIG. 4D) as well as triacylated lipopeptide (FIG. 4E). Incontrast, the diacylated lipopeptide FSL-1 was dependent on TLR2 andTLR6, but not TLR1 (FIG. 4F). All macrophage types responded to CpG ODN1826, a TLR9 agonist, confirming responsiveness of these cells to otherTLR stimuli (FIG. 4G). Other studies with macrophages from CD14^(−/−)mice showed that NA-LprG activity was dependent on CD14 (data notshown), consistent with observations that CD14 contributes to TLR2recognition of triacylated lipopeptides and glycolipids. The dependenceof NA-LprG and Mtb glycolipid signaling on TLR2/TLR1 and CD14 suggeststhat putative TLR2 agonist(s) associated with NA-LprG have recognitionrequirements similar to triacylated mycobacterial glycolipids, includingLM and LAM (and consistent with TLR2-dependent, TLR6-independentactivity of PIM6).

To directly identify molecules that non-covalently associate withNA-LprG, proteins were purified from M. smegmatis by and subjected toSDS-PAGE with silver stain, Pro-Q stain for carbohydrates followingperiodate oxidation (see Methods), or Western blot using a monoclonalanti-His₆ antibody or a polyclonal anti-M. bovis BCG antibody thatrecognizes many components of BCG and Mtb (FIG. 5). Silver stain andanti-His₆ Western of protein preparations showed an isolated band atapproximately 24 kDa representing NA-LprG, NA-LprG-V91W or NA-LprA (FIG.5A). The Pro-Q carbohydrate stain showed diffuse bands with apparentmolecular weights of 25-35 kDa, 14-18 kDa and 8-9 kDa (FIG. 5A, rightpanel), which correspond to the apparent molecular weights by SDS-PAGEof Mtb glycolipids LAM, LM and PIM, respectively (FIG. 5B). Thesecompounds are likely glycolipids because they were seen afterperoxidation but not on conventional silver stain, and glycolipids inthe PIM-LAM series resolve as broad bands based on heterogeneity of thearabinose and mannan components in each molecular species. Importantly,the bands corresponding to LAM and LM were more prominent with NA-LprGthan NA-LprG-V91W or LprA, suggesting that these glycolipids, which arepredominantly tri-acylated, are associated preferentially with NA-LprGwith little or no association with NA-LprG-V91W or NA-LprA. PIM wasassociated with all three proteins, possibly related to its existence inmultiple states of acylation, including diacylated as well astriacylated forms (below). The polyclonal anti-BCG antibody detectedNA-LprG and bands with apparent molecular weights consistent with theLAM and LM (FIG. 5); monoclonal antibody to LAM stained the highermolecular weight band (data not shown). These results provided evidencefor Mtb glycolipid association with NA-LprG but not NA-LprG-V91W orNA-LprA. Of note, LAM and LM share a common core of triacylated PIM(although other PIM acylation variants exist), suggesting that all ofthese mycobacterial glycolipids associate with NA-LprG and signalthrough TLR2/TLR1 via a shared structural motif.

To directly determine the molecular structures of small moleculesassociated with LprG and related proteins, we treated proteins withmethanol to denature and solubilize lipids. We then analyzed methanoleluates with nanoelectrospray ionization mass spectrometry to detectcompounds with a mass to charge (m/z) up to 2000. Methanol alone (FIG.6A) or methanol elutes of an unrelated protein Pab C (not shown) did notgive detectable ions. In contrast, both LprG (not shown) and NA-LprG(FIG. 6A) expressed in M. smegmatis yielded ions corresponding tomycobacterial phospholipids. Ions detected at m/z 851.4, 1013.5, 1175.5and 1413.7 corresponded in mass to [M-H]− of phosphatidylinositol,diacyl phosphatidylinositol monomannoside (PIM₁), diacyl PIM₂ andtriacyl PIM₂, respectively. Collision induced dissociation massspectrometry (CID-MS) analysis of these compounds yielded product ionsexpected from these assigned molecules, confirming the tentativeassignments of these compounds (FIG. 6A upper panels). For example,triacyl Ac₁PIM2 (m/z 1413.7) yielded products corresponding to the lossof mannose (m/z 1251), loss of acyl mannose (m/z 1013), loss of C16:0acyl (m/z 1157), loss of C19:0 fatty acyl (m/z 1115) and acylphosphoinositol dimannoside (m/z 803). Thus, analysis of compounds ofmolecular weight under 2000 mu showed that LprG binds at least fourstructurally related molecules, each of which is composed ofphosphatidylinositol as the core structure, but differ in the number ofmannose units and fatty acyl chains.

In comparative nanoelectrspray analysis of eluents of NA-LprG,NA-NA-LprA, the three diacylated compounds were detected in associationwith all three proteins (FIG. 6B-D), suggesting that both LprG and LprAbind diacylated ligands. Association of all three diacylated compoundswas decreased with NA-LprG-V91W, consistent with alteration of thehydrophobic putative binding pocket by this point mutation. Whilediacylated molecules were associated with both NA-LprG and NA-LprA, thetriacylated molecule Ac₁PIM2 was associated preferentially with NA-LprGand was not associated with NA-LprA or NA-LprG-V91W (FIG. 6E). Whilethese studies did not address triacylated glycolipids of highermolecular weight, these results demonstrate that LprG binds triacylatedAc₁PIM2 and suggest that LprG may also bind other Mtb glycolipids thatinclude the Ac₁PIM2 structure (e.g. LM and LAM).

To directly test the ability of NA-LprG to bind specific candidateglycolipids and deliver them for recognition by TLR2, we purifiedNA-LprG from E. coli (with little or no TLR2 activity) and incubated itwith preparations of mycobacterial glycolipids, including LM and LAMfrom M. smegmatis. After incubation with glycolipid, NA-LprG wasrepurified and tested for TLR2 agonist activity (FIG. 7). NA-LprG wasable to bind and deliver them for recognition by TLR2. In contrast,NA-LprA did not bind and deliver these glycolipids, and NA-LprG-V91Wbound them at reduced levels, consistent with prior evidence for theirrelatively specific association with NA-LprG (FIG. 5) suggesting thatdelivery by NA-LprG enhances the bioavailability or delivery of the TLR2agonists, or enhances their recognition by TLR2. We conclude thatNA-LprG is able to bind mycobacterial glycolipids and enhance theirrecognition by TLR2.

Since the preceding studies were performed with non-acylated variants ofthe lipoproteins, one question is how significant the glycolipid bindingsite and chaperoned TLR2 agonist are in terms of the total TLR2 agonistactivity of acylated LprG. Acylated LprG-V91W was found to besignificantly less potent than wild-type acylated LprG (FIG. 8). Theseresults suggest that alteration of the putative glycolipid binding sitemay reduce glycolipid binding or alter the types of glycolipids bound tofavor those with less TLR2 agonist activity (consistent with resultswith NA-LprG), and reduced binding of glycolipid TLR2 agonistssignificantly reduces TLR2 agonist activity of LprG. Thus, mutation ofthe putative glycolipid binding site reduces TLR2 agonist activity ofacylated LprG, indicating that TLR2 agonist activity of LprG reflectssignificant contributions by TLR2 agonists chaperoned by LprG as well asacylation of LprG itself.

Materials and Methods

Cloning and Expression of 6× Histidine (His₆) Tagged Proteins

LprA and NA-LprA were cloned previously. LprG was amplified from MtbH37Rv genomic DNA by PCR using the 5′ primerGCATATCCATATGCGGACCCCCAGACGCCACTG (SEQ ID NO: 3) and the 3′ primerGTACAAGCTTGCTCACCGGGGGCTTCG (SEQ ID NO: 4). A non-acylated (NA) variantof LprG was cloned by using a 5′ primer that excluded the signalsequence and changed the acylated cysteine to a methionine. NA-LprG wascloned with the following primers (underlined sequence is restrictionenzyme recognition site): 5′ gcaattccatatgtcgtcgggctc (SEQ ID NO: 5) and3′ gtacaagcttgctcaccgggggcttcg (SEQ ID NO: 6). Fusions of NA-LprA andNA-LprG were produced by digestion of the NA-Lpr constructs with NdeI,mscI, and HindIII, and ligating the 5′ fragment of NA-LprA with the 3′fragment of NA-LprG to make the A:G fusion protein. For the G:A fusion,the same digest was performed, and 5′ NA-LprG was fused with the 3′NA-LprA fragment. Site-directed mutagenesis of NA-LprG was performedusing the Quikchange site directed mutagenesis kit (Stratagene, 200519)with the following primers: 5′ gccgcgacgggaaactggaagctcacgctgggt (SEQ IDNO: 7) and 3′ acccagcgtgagcttccagtttcccgtcgcggc (SEQ ID NO: 8). Forexpression in M. smegmatis, constructs were digested with NdeI andHindIII (NEB, Ipswitch, Mass.) and ligated into the shuttle vector pVV16(provided by J. Belisle, Colorado State University, Fort Collins, Colo.)behind the constitutively active hsp60 promoter and in-frame with ac-terminal His₆ tag. For expression in E. coli Rosetta (EMD, cat#),constructs were digested with NdeI and HindIII and ligated with theexpression plasmid pET-22b(+) (Novagen) removing the pelB leadersequence, behind the IPTG-inducible T7 promoter and in frame with ac-terminal His₆ tag. All constructs were verified by sequencing andanalyzed using Clone Manager (SciEd software, Cary, N.C.). M. smegmatiswas transformed by electroporation with a Gene Pulser (Bio-Rad,Hercules, Calif.) set at 2.5 kV, 25 μF, and 800 Ohms. His₆-taggedproteins were expressed in M. smegmatis MC² 1-2C(R. Wilkinson, ImperialCollege, London, U.K.) cultivated in Middlebrook 7H9 broth (Difco,Lawrence, Kans.) supplemented with 1% casamino acids (Fisher,Pittsburgh, Pa., BP1424), 0.2% glycerol (Fisher G33-1), 0.2% glucose,and 0.05% Tween 80. Kanamycin was used at 30 μg/ml for selection of M.smegmatis. For expression in E. coli, chemically competent E. coliRosetta (EMD, cat#) were transformed according to the manufacturer'sprotocol. His₆-tagged proteins were expressed in E. coli Rosettacultivated in Lurie-Bertani broth (LB), induction of gene expression wasachieved by addition of 500 nM IPTG (Invitrogen, 15529-019) when cultureOD₆₀₀ was approximately 1.0, with 2-3 h subsequent growth at 37° C.before collection of the cells by centrifugation. Co-selection withAmpicillin (50 ug/ml) and Chloramphenicol (34 ug/ml) were used for E.coli Rosetta expression strains. Bacteria were isolated bycentrifugation at 6000×g for 20 min at 4° C.

Lysis and Purification of His₆-Tagged Proteins

Purification of NA-LprA and NA-LprG was accomplished as reportedpreviously. Cells were resuspended in lysis buffer (2.5 ml/liter ofbacterial culture) consisting of 50 mM NaH₂PO₄, 300 mM NaCl, 20 mMimidazole, pH 8.0, 2.5% protease inhibitor cocktail (Sigma P8849), 75U/ml benzonase (Novagen, Madison, Wis., 70664-3), and 2.5 mg lysozyme(Sigma L-3790) and incubated for 15 min at 37° C. Bacteria weredisrupted mechanically by 4 passages through a French press (2000 psi).Insoluble material was removed from the lysate by ultracentrifugation at100,000×g for 1 h at 4° C., and supernatant was incubated directly withNi-NTA beads (Qiagen, Valencia, Calif., 1018244) for 2-4 h at 4° C.Ni-NTA beads were transferred to polypropylene columns, washed 3× with25 volumes of wash buffer (50 mM NaH₂PO₄, 1 M NaCl, 20 mM imidazole, 10%glycerol, pH 8.0), and bound protein was dissociated with elution buffer(50 mM NaH₂PO₄, 300 mM NaCl, 450 mM imidazole, pH 8.0). To prepare foranion exchange chromatography, sample was desalted into 20 mM Tris, pH8.0 using PD-10 columns (GE Healthcare, Uppsala, Sweden 17-085-01).Samples were subjected to anion exchange chromatography using quaternaryammonium columns (GE Healthcare, 17-5053-01), and eluted with theaddition of NaCl in the following steps: 50, 150, 200, 1000 mM. Presenceand purity of desired protein was verified by SDS-PAGE and visualized assingle bands by silver stain and anti-His₆ Western blot; yields wereestimated by BCA protein assay (Pierce, Rockford, Ill., 23225). Materialeluted by 50 and 150 mM NaCl was used for all experiments.

Culture of Mtb H37Ra

Mtb strain H37Ra (ATCC 25177) was cultured with shaking at 37° C. tolate log phase growth (2.5 weeks) in Mtb 7H9 broth (4.7 g/17H9 (Difco271310), 5 ml/l glycerol, 0.5 ml/l Tween-80 (Sigma, St. Louis, M0,P4780) supplemented with 10% albumin/dextrose/catalase (BD, FranklinLakes, N.J. 212352). Bacilli were harvested by centrifugation at 5,000×gfor 20 min at 4° C. 100 ml of late log phase culture was harvested bycentrifugation and resuspended in 5 ml volume with culture supernatantand stored at −80° C.

Crystallization and Determination of NA-LprG Structure

Visualization of the crystal structure and design of mutation studieswas performed using Deepview 4.0. Predicted structures of LprA and thefusions between LprA and LprG were generated using SWISS-MODEL.Molecular graphics images were produced using the UCSF Chimera packagefrom the Resource for Biocomputing, Visualization, and Informatics atthe University of California, San Francisco (NIH P41 RR-01081).

Charging of E. coli-Derived Proteins

For charging of E. coli-expressed proteins with M. smegmatis or MtbH37Ra sonicate, thawed aliquots of cells suspended in 5 ml of mediumwere sonicated at amplitude 1 for four 15 minute bursts in ice waterwith a temperature cut-off of 40° C. Insoluble material was removed fromthe sonicate by centrifugation at 10,000×g for 20′ at 4° C. Thesupernatant was incubated with 300-500 μg of protein for 3 h, rocking at37° C. Charged proteins were re-purified by Ni-NTA affinity andanion-exchange chromatography. Proteins purified from E. coli weresimilarly charged with purified glycolipids or culture filtrate. Inthese experiments, 100 μg of E. coli-expressed, purified His₆-taggedprotein was incubated with 50 μg of purified glycolipid or 100 μl oflysis buffer for 3 h at 37° C. The His₆-tagged proteins were thenre-purified by Ni-NTA affinity and anion exchange chromatography andused in bioassays.

SDS-PAGE and Visualization of Purified Proteins and Glycolipids

Gels (13% acrylamide) were cast and run using a Tris-HCl buffer system.Proteins were visualized with Silver Stain Plus (BioRad), andcarbohydrates (including glycolipids) were visualized with Pro-Q 3000(Molecular Probes). For Western analysis was performed with transfer ofmaterial to a PVDF membrane, which was blocked with 5% Milk in PBSsupplemented with 0.1% Tween-20 (PBST) for 1 h and incubated overnightat 4° C. with antibody (rabbit polyclonal anti-BCG, 1:30,000, DAKO;mouse monoclonal anti-His₆, 1:1000, Santa Cruz; or mouse monoclonalanti-LAM antibody CS-35, provided by Colorado State University under theNIH TBVTRM contract). Blots were then washed three times in PBST,incubated for 2 h at room temperature with secondary goat anti-rabbit orhorse anti-mouse antibodies diluted 1:2000 in PBST, and washed threetimes in PBST. Reactive bands were visualized with chemiluminescence (GEHealthcare).

Mass spectrometry and Identification of Ligands of NA-LprG

Mammalian Cell Culture

Unless otherwise specified, incubations with eukaryotic cells wereperformed at 37° C. in 5% CO₂ atmosphere. Standard medium was DMEM(Hyclone, Logan, Utah, ASK30773) supplemented with 10% heat-inactivatedFCS, 50 μM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, pH7.4, and penicillin/streptomycin (Hyclone). Stimulation medium wasstandard medium with serum concentration reduced to 0.2% FBS. FemaleC57BL/6J mice (8-16 weeks old) were obtained from the JacksonLaboratory, housed under specific pathogen-free conditions and used toproduce macrophages. TLR1^(−/−), TLR2^(−/−) and TLR6^(−/−) mice weregenerously provided by Shizuo Akira (Research Institute for MicrobialDisease, Osaka University, Osaka, Japan) and were back-crossed toC57BL/6J mice a minimum of eight times. CD14 knockout mice(B6.129S-Cd14^(tm1Frm)/J) were obtained from the Jackson Laboratory,maintained under specific pathogen-free conditions and used to producemacrophages. CD14 knockout mice were compared to C57BL/6J mice and F2hybrids of C57BL/6J and 129sv. Bone marrow cells were cultured for 7-12d in standard medium supplemented with 25% LADMAC cell-conditionedmedium. HEK293 cells stably expressing TLR2-YFP (HEK293.TLR2) wereproduced previously. HEK293 cells (ATCC CRL-1573) were stablytransfected with the empty vector to produce a control HEK293.pcDNA3cell line. Transfected HEK293 cell lines were maintained in HEK medium(DMEM supplemented with 10% heat-inactivated FCS (HyClone)) supplementedwith ciprofloxacin (10 μg/ml) and geneticin (500 μg/ml).HEK293.TLR2-CD14 line was also purchased from Invivogen (Invivogen, cat#293-htlr2cd14, San Diego, Calif. 92121). These cells were cultured inHEK medium supplemented with 100 ug/ml Normocin, 100 ug/ml Hygromycin B,and 10 ug/ml Blasticidin.

Cytokine ELISAs

HEK293 cells were incubated in 96-well plates (20,000 cells/well) for5-8 h in 90 μl of appropriate HEK medium and then for an additional 16 hwith or without TLR2 agonist. Supernatant IL-8 concentration wasquantified by ELISA (R&D, Minneapolis, Minn., DY208). Bonemarrow-derived macrophages were incubated overnight at 100,000cells/well in standard medium and then for 12 h in stimulation mediumwith or without agonist. Supernatants were collected and stored at−80′C. TNF-alpha in the supernatant was quantified by ELISA (BDBiosciences #558874, R&D DY410). The following synthetic TLR agonistswere also used: Ultrapure E. coli LPS (Invivogen, San Diego, Calif.,tlrl-pelps), FSL-1 (Invivogen, tlrl-fsl) and Pam₃CSK₄ (Invivogen,tlrl-pms).

Having described the invention, the following is claimed:
 1. An adjuvantcombination that stimulates an immune activation and/or responsecomprising: at least one of a TLR2 agonist or a TLR4 agonist and apurified non-acylated or acylated Myobacterium tuberculosis LprGlipoprotein.
 2. An immune stimulating complex comprising: at least oneof a TLR2 agonist or a TLR4 agonist and purified non-acylated oracylated Myobacterium tuberculosis LprG lipoprotein, the LprGlipoprotein being present in an amount effective to chaperone the TLR2agonist or the TLR4 agonist to an immune receptor.